1. Field of the Invention
The present invention relates generally to the fields of pancreatic islet isolation and medical therapy for diabetes. More specifically, the present invention relates to improved procedures for isolating pancreatic islets that better protect the islets during the isolation process. In order to screen for new drugs to treat type 2 diabetes, or to prepare islets for transplantation for type 1 diabetes, isolated islets must be protected from toxic molecules that form during the isolation process.
2. Description of the Related Art
Early in the course of type 2 diabetes, pancreatic xcex2-cell function is sufficient such that in many patients, oral hypoglycemic agents are adequate to compensate for increased insulin resistance (1). As type 2 diabetes progresses, however, xcex2-cells lose their capacity to produce sufficient amounts of insulin to control the blood glucose level and patients become increasingly hyperglycemic (2). It has been suggested that the hyperglycemia itself may cause damage to xcex2-cells (3-5). The exact mechanism by which a n increased concentration of glucose may affect xcex2-cells, however, is not completely elucidated.
One metabolite of glucose that has been proposed to mediate adverse effects of hyperglycemia is glucosamine (2-amino-2-deoxyglucose) (6,7). Glucosamine is a product of glucose metabolism and is synthesized from fructose-6-phosphate by the apparently unique and rate limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFAT) (8-10). This metabolic step provides the substrate UDP-N-acetylglucosamine (UDP-GlcNAc) for glycoprotein synthesis. Quantitatively, most glycosylation occurs on proteins destined for export from the cell or for the plasma membrane of the cell. In eukaryotic cells, however, there is a cytoplasmic form of glycosylation that involves O-linkage of the monosaccharide, N-acetylglucosamine (GlcNAc), to proteins at serine or threonine residues (11-19). An enzyme responsible for this form of protein modification, O-linked N-acetylglucosamine transferase (OGT), has recently been characterized, and its cDNA has been cloned (20). Studies on the tissue distribution of O-GlcNAc transferase have indicated that O-linked N-acetylglucosamine transferase mRNA, although ubiquitous, is particularly abundant in the pancreatic xcex2-cell (21, 22).
Interestingly, an analog of N-acetylglucosamine called streptozotocin (STZ) has been used to create animal models of diabetes (27). A nitrosourea group is present at a position that corresponds to the acetate in N-acetylglucosamine (FIG. 6). A single dose of 50-100 mg/kg of streptozotocin administered to a rat causes death of most of the xcex2-cells and development of diabetes. Very recently, streptozotocin has been shown to act by inhibiting the enzyme O-GlcNAc-selective N-acetyl-xcex2-D-glucosaminidase (O-GlcNAcase), which cleaves O-linked N-acetylglucosamine off protein (21, 22). These data suggest why the xcex2-cell, with its elevated level of O-GlcNAc transferase, may be particularly vulnerable to streptozotocin.
Supporting this idea is a recent report that xcex2-cell O-linked protein glycosylation is involved in xcex2-cell apoptosis in vivo since administration of streptozotocin to rats prior to the induction of hyperglycemia results in irreversible increases in xcex2-cell O-glycosylation and subsequent xcex2-cell apoptosis (23). In addition, both glucose and streptozotocin stimulate O-glycosylation of a 135 kD protein in isolated islets in vitro, although the effect did not seem as dramatic as the reported in vivo observations (24).
The prior art is deficient in the lack of effective means of blocking the formation and action of molecules that are toxic to the xcex2-cells of the pancreatic islets when the islets are being isolated from the pancreas. The prior art is also deficient in a means by which to determine if pancreatic xcex2-cells are traumatized by toxic molecules such as nitric oxide or streptozotocin-like compounds during the isolation process. Additionally, the prior art is deficient in a means by which to test diabetes drugs to see if they affect glucose toxicity to xcex2-cells by acting through the O-linked protein glycosylation pathway that glucose, analogues of glucosamine, and streptozotocin affect. The present invention fulfills these long-standing needs and desires in the art.
The present invention demonstrates that during standard islet isolation (which has been performed for the last 30 years), pancreatic xcex2-cells are affected adversely by nitric oxide and/or streptozotocin-like molecules that form during the isolation process. In order to prevent nitric oxide toxicity or streptozotocin-like toxicity to the islets during the isolation process, the islets need to be isolated in the presence of high glucose and glutamine, glucosamine, N-acetylglucosamine, or other agents that act through the N-acetylglucosamine (GlcNAc) pathway. Such toxicity can further be reduced by the addition of nitric oxide synthase (NOS) inhibitors and/or nitric oxide (NO) scavengers. The present invention also discloses that streptozotocin, glucose, and glucosamine all increase the O-glycosylation of protein in xcex2-cells, and that assaying for modification of this protein can allow a determination to be made as to islet viability as well as which potential diabetes drugs might be likely candidates for acting through the O-linked glycosylation pathway.
Glucose, glucosamine, and streptozotocin stimulate O-glycosylation of a single 135 kD xcex2-cell protein in isolated rat islets. The effect of glucose, but not that of glucosamine or streptozotocin was blocked by inhibition of glutamine:fructose-6-phosphate amidotransferase, suggesting that glucose acts through the glucosamine pathway to provide more UDP-N-acetylglucosamine substrate for O-linked N-acetylglucosamine transferase to attach to p135. The effect of glucose on p135 O-glycosylation was reversible while that of streptozotocin was not, indicating that streptozotocin irreversibly inactivated xcex2-cell O-GlcNAcase. Experiments performed in clonal xcex2-cell lines demonstrated an inability of xcex2-cells grown in culture to regulate increased p135 O-glycosylation in response to glucose, glucosamine, or streptozotocin, indicating a fundamental difference between xcex2-cells grown in culture and actual xcex2-cells in islets. Together, these data provide strong evidence that glucose and streptozotocin act through a common pathway of p135 O-glycosylation that is present only in primary xcex2-cells in islets. The observation that both glucose and streptozotocin stimulate p135 O-glycosylation provides a possible mechanism by which hyperglycemia may cause streptozotocin-like effects in xcex2-cells and thus contribute to the development of type 2 diabetes.
In one embodiment of the present invention, there is provided a method of isolating pancreatic islets so as to protect xcex2-cells from toxicity of nitric oxide and/or streptozotocin-like molecules generated during the isolation process by isolating the islets in the presence of compound(s) that acts through the glucosamine pathway.
In another embodiment of the present invention, islets are isolated in the presence of high glucose (6-300 mM) and glutamine (0.001-300 mM) in order to protect xcex2-cells from toxicity of nitric oxide and/or streptozotocin-like molecules.
In another embodiment of the present invention, islets are isolated in the presence of glucosamine (0.001-300 mM) in order to protect xcex2-cells from toxicity of nitric oxide and/or streptozotocin-like molecules.
In another embodiment of the present invention, islets are isolated in the presence of N-acetylglucosamine (0.001-300 mM) in order to protect xcex2-cells from toxicity of nitric oxide and/or streptozotocin-like molecules.
In another embodiment of the invention, islets are isolated in the presence of nitric oxide (NO) inhibitors such as nitric oxide synthase inhibitors and/or nitric oxide scavengers in order to prevent the formation of nitric oxide and/or streptozotocin-like molecules and protect xcex2-cells.
In another embodiment of the invention, islets are isolated in the presence of high glucose (6-300 mM) and glutamine (0.001-300 mM) and nitric oxide inhibitors in order to protect xcex2-cells from toxicity of nitric oxide and/or streptozotocin-like molecules.
In another embodiment of the invention, islets are isolated in the presence of glucosamine (0.001-300 mM) and nitric oxide inhibitors in order to protect xcex2-cells from toxicity of nitric oxide and/or streptozotocin-like molecules.
In another embodiment of the present invention, islets are isolated in the presence of N-acetylglucosamine (0.001-300 mM) and nitric oxide inhibitors in order to protect xcex2-cells from toxicity of nitric oxide and/or streptozotocin-like molecules.
In another embodiment of the present invention, there is provided a method to determine if nitric oxide and/or streptozotocin-like molecules have damaged islets. Islets are pre-incubated with low glucose (0-6 mM), and then stimulated with low glucose (0-6 mM), high glucose (6-300 mM), glucosamine (0.001-300 mM), N-acetylglucosamine (0.001-300 mM), or streptozotocin (0.001-300 mM). Islet proteins are then analyzed for protein O-glycosylation, wherein no change in O-glycosylation of a 135 kD protein indicates said islets have been damaged with nitric oxide and/or streptozotocin-like molecules.
In another embodiment of the present invention, there is provided a method of assaying isolated islets or beta-cells to determine if candidate diabetes drugs might be effective in treating diabetes. Islets or beta-cells are isolated according to the methods described above, pre-incubated with low glucose (0-6 mM), and then either stimulated with low glucose (0-6 mM), high glucose (6-300 mM), glucosamine (0.001-300 mM), N-acetylglucosamine (0.001-300 mM), or streptozotocin (0.001-300 mM), with or without the drug(s) of interest present. Islet or beta cell proteins are then analyzed for protien O-glycosylation, wherein inhibition of O-glycosylation on a 135 kD protein in the presence of said candidate drug indicates said candidate diabetes drug might be effective in treating diabetes.